Novel saturation transfer difference (STD) NMR approaches to understand biologically relevant protein-carbohydrate interactions

Monaco, Serena (2018) Novel saturation transfer difference (STD) NMR approaches to understand biologically relevant protein-carbohydrate interactions. Doctoral thesis, University of East Anglia.

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Saturation transfer difference (STD) NMR spectroscopy is a powerful NMR technique extensively used to obtain epitope maps of ligands binding to biologically relevant protein receptors. This allows to reveal semi-quantitative structural details of the interaction, which is key to direct lead optimization efforts in drug discovery. However, it does not give information about the nature of the amino acids surrounding the ligand in the binding pocket.
In this thesis, the main effort has been put to develop two novel implementations of STD NMR, aimed at elucidating the surroundings of the ligand (i.e., the amino acids lining the binding pocket, or an adjacent bound ligand) in biologically relevant complexes. First, we report the development of the novel “DiffErential EPitope mapping STD NMR” (DEEP‐STD NMR), a method producing differential epitope maps through i) differential frequency and/or ii) differential solvent (D2O/H2O) STD NMR experiments. These two approaches provide complementary information on the architecture of the binding pocket. The second novel method we propose is “Inter-ligand STD NMR” (IL-STD NMR), which relies on on-ligand differential frequency STD NMR to detect contacts between ligands bound to adjacent sites of a receptor.
These novel STD NMR methodologies, in combination with traditional STD NMR and computational tools, have been applied to the study of two systems: the interactions of Cholera Toxin subunit B (CTB) with a set of promising inhibitors; and the interactions of an intramolecular trans-sialidase from Ruminococcus gnavus, a gut microbiota symbiont, with a set of mucin-related sialylated ligands. In the first study, we discovered the existence of a hitherto unknown binding subsite in the GM1 binding site of CTB. In the second study, we provided the first 3D molecular model of a Michaelis complex for an IT-sialidase.
In both cases, we demonstrate that our newly developed approaches increase the level of resolution of STD NMR, widening its potential to impact the field of ligand design for biologically relevant receptors.

Item Type: Thesis (Doctoral)
Faculty \ School: Faculty of Science > School of Pharmacy
Depositing User: Jackie Webb
Date Deposited: 05 Oct 2018 13:22
Last Modified: 05 Oct 2018 13:22

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